SmartPhos command line
2026-02-03
1 Creating an R object from Spectronaut output files
[Skip for now]
filetable <- read.table(file="/Users/shubhamagrawal/Documents/work/compressed/DIA/fileTable.txt", header = TRUE)
mae <- readExperimentDIA(fileTable = filetable,
annotation_col = c("treatment", "timepoint", "replicate",
"sampleType"))2 Use the created object
mae <- readRDS("data/maeObjNEW.Rds")
mae## A MultiAssayExperiment object of 2 listed
## experiments with user-defined names and respective classes.
## Containing an ExperimentList class object of length 2:
## [1] Phosphoproteome: SummarizedExperiment with 40517 rows and 138 columns
## [2] Proteome: SummarizedExperiment with 8518 rows and 79 columns
## Functionality:
## experiments() - obtain the ExperimentList instance
## colData() - the primary/phenotype DataFrame
## sampleMap() - the sample coordination DataFrame
## `$`, `[`, `[[` - extract colData columns, subset, or experiment
## *Format() - convert into a long or wide DataFrame
## assays() - convert ExperimentList to a SimpleList of matrices
## exportClass() - save data to flat files3 Preprocessing the assay, basic visualization, PCA
se <- mae[["Phosphoproteome"]]
colData(se) <- colData(mae[, colnames(se)])
se## class: SummarizedExperiment
## dim: 40517 138
## metadata(0):
## assays(1): Intensity
## rownames(40517): s1 s2 ... s40516 s40517
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(138): FullProteome_1stCtrl_0min_rep2
## FullProteome_1stCtrl_0min_rep3 ... Phospho_HGF_40min_rep1
## Phospho_HGF_6h_rep1
## colData names(6): treatment timepoint ... sample sampleNameplotIntensity(se[, se$sampleType == "Phospho"], color = "replicate") + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
) 
newSE <- preprocessPhos(seData = se, transform = "log2",
normalize = TRUE, impute = "QRILC")## Warning in fun(libname, pkgname): mzR has been built against a different Rcpp version (1.0.12)
## than is installed on your system (1.1.0). This might lead to errors
## when loading mzR. If you encounter such issues, please send a report,
## including the output of sessionInfo() to the Bioc support forum at
## https://support.bioconductor.org/. For details see also
## https://github.com/sneumann/mzR/wiki/mzR-Rcpp-compiler-linker-issue.## Imputing along margin 2 (samples/columns).newSE## class: SummarizedExperiment
## dim: 13081 59
## metadata(0):
## assays(2): Intensity imputed
## rownames(13081): s1 s4 ... s40511 s40514
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(59): Phospho_1stCtrl_0min_rep2 Phospho_1stCtrl_0min_rep3 ...
## Phospho_HGF_40min_rep1 Phospho_HGF_6h_rep1
## colData names(6): treatment timepoint ... sample sampleNameplotIntensity(newSE, color = "replicate") + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
) 
plotMissing(newSE) + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
) 
4 Perform PCA
pca <- stats::prcomp(t(assays(newSE)[["imputed"]]), center = TRUE, scale. = TRUE)p <- plotPCA(pca = pca, se = newSE,
color = "treatment",
shape = "replicate")
p$layers[[1]]$aes_params$size <- 3
p$layers[[1]]$aes_params$stroke <- 1.2
p + coord_equal() + theme(aspect.ratio = 1, legend.position = "right",
axis.text = element_text(color = "#000000")) +
guides(
shape = guide_legend(order = 1),
color = guide_legend(order = 2)
)
5 rep1 samples need to be removed or perform batch correction
newSE <- preprocessPhos(seData = se, transform = "log2",
normalize = TRUE, impute = "QRILC",
removeOutlier = "rep1")## Imputing along margin 2 (samples/columns).newSE## class: SummarizedExperiment
## dim: 17035 32
## metadata(0):
## assays(2): Intensity imputed
## rownames(17035): s1 s4 ... s40513 s40514
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(32): Phospho_1stCtrl_0min_rep2 Phospho_1stCtrl_0min_rep3 ...
## Phospho_HGF_40min_rep2 Phospho_HGF_40min_rep3
## colData names(6): treatment timepoint ... sample sampleNamepca <- stats::prcomp(t(assays(newSE)[["imputed"]]),
center = TRUE, scale. = TRUE)p <- plotPCA(pca = pca, se = newSE,
color = "treatment",
shape = "replicate")
p$layers[[1]]$aes_params$size <- 3
p$layers[[1]]$aes_params$stroke <- 1.2
p + coord_equal() + theme(aspect.ratio = 1, legend.position = "right",
axis.text = element_text(color = "#000000")) +
guides(
shape = guide_legend(order = 1),
color = guide_legend(order = 2)
)
p <- plotHeatmap(type = "Top variant", newSE, top = 30, annotationCol = c("replicate", "treatment")) 
g <- p$gtable
# Find row names grob
row_names <- which(g$layout$name == "row_names")
g$grobs[[row_names]]$gp <- gpar(fontsize = 8, fontface = "bold")
# Same for columns
col_names <- which(g$layout$name == "col_names")
g$grobs[[col_names]]$gp <- gpar(fontsize = 10)
grid.newpage()
grid.draw(g)
6 Differential expression
dea <- performDifferentialExp(se = newSE,
assay = "imputed",
method = "limma",
condition = "treatment",
reference = "1stCtrl",
target = "H.E")p <- ggplot(dea$resDE, aes(x = pvalue)) +
geom_histogram(fill = "grey", col = "blue", alpha=0.7) +
ggtitle("P value histogram") + theme_classic() +
theme(plot.title = element_text(hjust = 0.5))
p## `stat_bin()` using `bins = 30`. Pick better value `binwidth`.
pFilter = 0.01
plotVolcano(dea$resDE, pFilter = 0.01, fcFilter = 1) + theme_classic() +
geom_hline(yintercept = -log10(as.numeric(pFilter)), color = "brown",
linetype = "dashed") +
annotate(x = 3.0, y = -log10(as.numeric(pFilter)) - 0.20,
label = paste0("P-value = ", as.numeric(pFilter)),
geom = "text", size = 3.5, color = "brown") 
plotVolcanoDEA(dea$resDE, fcFilter = 1, pFilter = 0.01, usePadj = FALSE)
dea$resDE## # A tibble: 17,035 × 11
## ID log2FC stat pvalue padj UniprotID Gene Position Residue Sequence
## <I<c> <dbl> <dbl> <dbl> <dbl> <chr> <chr> <chr> <chr> <chr>
## 1 s176… -3.50 -35.2 1.13e-12 1.92e-8 Q3MIN7 RGL3 573 S PAGSPPA…
## 2 s2851 8.40 27.4 1.70e-11 1.08e-7 O60885 BRD4 470 S EPVVAVS…
## 3 s128… 7.12 27.1 1.91e-11 1.08e-7 Q06124 PTPN… 580 Y REDSARV…
## 4 s4880 8.35 22.1 1.72e-10 7.32e-7 O95870 ABHD… 32 T APASVPE…
## 5 s398… 6.86 20.7 3.61e-10 1.21e-6 Q9Y4K1 CRYB… 363 T SSAQADC…
## 6 s129… 3.33 20.4 4.25e-10 1.21e-6 Q07889 SOS1 1134 S PHGPRSA…
## 7 s5284 2.69 19.7 6.13e-10 1.34e-6 P04920 SLC4… 243 S QERRRIG…
## 8 s383… 6.82 19.6 6.28e-10 1.34e-6 Q9UPY5 SLC7… 26 S NVNGRLP…
## 9 s582 -2.29 -19.0 8.77e-10 1.58e-6 O00401 WASL 256 Y RETSKVI…
## 10 s668 2.58 18.9 9.56e-10 1.58e-6 O00515 LAD1 38 S RRRHRNL…
## # ℹ 17,025 more rows
## # ℹ 1 more variable: site <chr>intensityBoxPlot(se = dea$seSub, id = 's4971', symbol = "EGFR_S991")
7 Time series clustering
set.seed(12345)
newSEts <- newSE[ , newSE$treatment == "H.E"]
assayMat <- SummarizedExperiment::assay(newSEts)
exprMat <- lapply(unique(newSEts$timepoint), function(tp) {
rowMedians(assayMat[,newSEts$timepoint == tp])
}) %>% bind_cols() %>% as.matrix()## New names:
## • `` -> `...1`
## • `` -> `...2`
## • `` -> `...3`
## • `` -> `...4`rownames(exprMat) <- rownames(newSEts)
colnames(exprMat) <- unique(newSEts$timepoint)
sds <- apply(exprMat,1,sd)
varPer <- 80
exprMat <- exprMat[order(sds, decreasing = TRUE)[seq(1, varPer/100*nrow(exprMat))], ]
# only center when it's for expression
exprMat <- mscale(exprMat)
# remove NA values
exprMat <- exprMat[complete.cases(exprMat), ]
tsc <- clusterTS(x = exprMat, k = 5, pCut = 0.6)tsc$plot + theme(
axis.text.x = element_text(size = 10), axis.text = element_text(color = "#000000"), ) 
8 Enrichment analysis
genesetPath <- system.file("shiny-app/geneset", package = "SmartPhos")
inGMT1 <- piano::loadGSC(paste0(genesetPath, "/Cancer_Hallmark.gmt"),
type="gmt")
resTab <- enrichDifferential(dea = dea$resDE, type = "Pathway enrichment",
gsaMethod = "PAGE", geneSet = inGMT1,
statType = "stat", nPerm = 200, sigLevel = 0.05,
ifFDR = FALSE)## Running gene set analysis:## Checking arguments...done!## Final gene/gene-set association: 1218 genes and 50 gene sets## Details:## Calculating gene set statistics from 1218 out of 4183 gene-level statistics## Removed 3168 genes from GSC due to lack of matching gene statistics## Removed 0 gene sets containing no genes after gene removal## Removed additionally 0 gene sets not matching the size limits## Loaded additional information for 50 gene sets## Calculating gene set statistics...done!
## Calculating gene set significance...done!
## Adjusting for multiple testing...done!resTab## Name Gene Number Stat p.up
## 1 HALLMARK_MITOTIC_SPINDLE 145 2.6658 0.0038407
## 2 HALLMARK_APICAL_SURFACE 10 1.7214 0.0425930
## 3 HALLMARK_MYC_TARGETS_V1 112 -1.6775 0.9532800
## 4 HALLMARK_MYOGENESIS 43 -1.7676 0.9614300
## 5 HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION 29 -1.9409 0.9738700
## 6 HALLMARK_G2M_CHECKPOINT 116 -2.0758 0.9810400
## 7 HALLMARK_KRAS_SIGNALING_DN 14 -2.5684 0.9948900
## 8 HALLMARK_HEDGEHOG_SIGNALING 10 -3.1079 0.9990600
## p.up.adj p.down p.down.adj Number up Number down
## 1 0.19203 0.99616000 0.996160 83 62
## 2 0.99906 0.95741000 0.976950 7 3
## 3 0.99906 0.04672300 0.389360 53 59
## 4 0.99906 0.03856600 0.385660 17 26
## 5 0.99906 0.02613400 0.326670 9 20
## 6 0.99906 0.01895800 0.315970 50 66
## 7 0.99906 0.00510840 0.127710 2 12
## 8 0.99906 0.00094225 0.047112 1 9inGMT2 <- piano::loadGSC(paste0(genesetPath, "/KEGG_pathways.gmt"),
type="gmt")
clustEnr <- clusterEnrich(clusterTab = tsc$cluster, se = newSE,
inputSet = inGMT2, filterP = 0.01, ifFDR = FALSE)clustEnr$plot + theme_classic()
9 Kinase activity inference
First on the differential expression analysis results
netw <- getDecouplerNetwork("Homo sapiens")
scoreTab <- calcKinaseScore(dea$resDE, netw, statType = "stat", nPerm = 100)
plotKinaseDE(scoreTab, nTop = 10, pCut = 0.05)
clusterData <- tsc$cluster[tsc$cluster$cluster == "cluster1",]
allClusterFeature <- clusterData %>%
distinct(feature, .keep_all = TRUE) %>% .$feature
allClusterSite <- data.frame(rowData(newSEts)[allClusterFeature, "site"])
allClusterSite$feature <- allClusterFeature
clusterData <- clusterData %>%
left_join(allClusterSite, by = "feature") %>%
rename(site = "rowData.newSEts..allClusterFeature...site..")
scoreTab <- calcKinaseScore(clusterData, netw, statType = "stat", nPerm = 100)
plotKinaseTimeSeries(scoreTab, pCut = 0.05, clusterName = "cluster1")10 Phosphosites pattern
newSEts <- addZeroTime(newSE, condition = "treatment", treat = "H.E",
zeroTreat = "1stCtrl",
timeRange = c("20min","40min","100min"))
rd <- as.data.frame(rowData(newSEts))
timerange <- unique(newSEts$timepoint)
sites <- c("MET_Y1234", "MET_Y1235", "MET_1003", "Shc1_Y427", "AKT3_Y312", "RPS6KB1", "EGFR_Y1092", "EGFR_Y1172", "SOS1_S1134", "MAP2K2_S226", "MAPK3_T202", "MAPK1_T190", "EGFR_Y1197", "ERBB2_S998","GAB1_Y659","RPS6_S244")
for (i in sites) {
# condition to check if that site is present in row data
if (i %in% rd$site) {
# condition to check if assay doesn't have NAs (imputed assays are not allowed)
if (!is.na(assay(newSEts)[rownames(rd[rd$site==i,]), ][1])) {
p <- plotTimeSeries(newSEts, type = "expression",
geneID = rownames(rd[rd$site==i,]),
symbol = i, condition = "treatment",
treatment = "H.E", timerange = timerange) +
theme(axis.text = element_text(color = "#000000", size = 20),
axis.text.x = element_text(angle = 0, vjust = 0, hjust = 0.5, size = 20),
axis.title = element_text(size=20),
plot.title = element_text(size=30)) +
scale_x_continuous(breaks = c(0,20,40,60,80,100))
p$layers[[1]]$aes_params$size <- 6
p$layers[[1]]$mapping$colour <- NULL
p$layers[[1]]$aes_params$colour <- "#c1191f"
p$layers[[2]]$aes_params$linewidth <- 1
p$layers[[2]]$mapping$colour <- NULL
p$layers[[2]]$aes_params$colour <- "#555555"
print(p)
}
}
}
